Method for modifying transglutaminases from microorganisms

ABSTRACT

The present invention relates to a method for designing and preparing mutant transglutaminases on the basis of the three-dimensional structure of MTG derived from  Streptoverticillium mobaraense  (MTG), and the mutant MTG thus prepared. The present invention provides a method for modifying MTG on the basis of the three-dimensional structure, and transglutaminase having reactivity on the substrate improved by the method. In the present invention, the binding site of MTG for the substrate is extrapolated based on the three-dimensional structure obtained by X-ray crystal structure analysis of MTG crystals, and the mutant transglutaminases are designed and produced by replacing, inserting or deleting amino acid residues positioned at the substrate-binding site of the transglutaminase.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a method for designing and preparing mutant transglutaminase on the basis of the three-dimensional structure of transglutaminase derived from Streptoverticillium mobaraense (hereinafter referred to as “MTG”) determined by X-ray crystal structure analysis techniques, and the mutant MTGs thus prepared. MTG is widely used for processing foods or the like because it forms a gelatinus substance by forming crosslinking between proteins. The mutant MTG improved in the transglutaminase activity and heat stability contributes to reduce the required amount thereof, and also the mutant MTG having modified substrate specificity and optimum pH will allow the application of the enzyme to a new field.

[0002] Transglutaminase is an enzyme which catalyzes the acyl transfer reaction of γ-carboxamide group existing in a peptide chain of a protein. By reacting a protein with this enzyme, ε-(γ-Glu)-Lys crosslinking reaction and replacement reaction of Gin with Glu by deamidation may occur.

[0003] Transglutaminases from animals and those from microorganisms are so far known. The former is a Ca²⁺-dependent enzyme and it is widely distributed in organs, skin and blood of animals. This includes, for example, guinea pig liver transglutaminase [K. Ikura et al., Biochemistry, Vol. 27, p. 2898 (1988)], human epidermal keratin cell transglutaminase [M. A. Phillips et al., Proc. Natl. Acad. Sci., USA, Vol. 87, p. 9333 (1990)] and human blood coagulation factor XIII [A. Ichinose et al., Biochemistry, Vol. 25, p. 6900 (1990)]. As for the latter, the Ca²⁺-independent ones were found in Streptoverticillium bacteria such as Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852 and Streptoverticillium mobaraense IFO 13819. Among these enzymes, the transglutaminase found in a culture supernatant of a variant of Streptoverticillium mobaraense is referred to as MTG (Microbial Transglutaminase). Also, a Ca²⁺-independent transglutaminase was found in Streptomyces lydicus NRRL B-3446 (JP-Kokai No. 10-504721).

[0004] MTG is a monomeric protein composed of 331 amino acids and having a molecular weight of about 38,000 [Journal of Biological Chemistry, vol. 268, p. 11565 (1993)]. A method for producing active MTG by secretion expression using E. coli or yeast was reported (JP-Kokai No. Hei 5-199883). There has also been reported a method for producing the same wherein MTG is expressed as a protein inclusion body in E. coli, then the inclusion body is solubilized with a protein denaturing agent and it is then regenerate through removing the denaturing agent to obtain active MTG (JP-Kokai No. 6-30771).

[0005] Unlike transglutaminases from animals, the transglutaminases from microorganisms, such as MTG, are Ca²⁺-independent. They are therefore utilized for the production of gelled foods such as jellies, as well as yogurts, cheeses or cosmetics in gel form, and also for the improvement of the quality of meats (JP-Kokai No. 64-27471). They are highly useful in the industry in that they are used as materials for heat stable microcapsules or as carriers for immobilized enzymes.

[0006] As for the conditions of the enzymatic reaction, for example, gelatinized foods cannot be gelled when the enzymatic reaction time is short, on the contrary, they become too hard to be the commercial products when the enzymatic reaction time is excessively long. Therefore, when MTG is used for producing gelatinized foods such as jellies; yogurts; cheeses; or cosmetics in gel form; or for improving the quality of meats, the concentration of the substrate and enzyme, reaction temperature and reaction time are controlled suitably for the intended product. However, as the foods and reagents produced by using MTG became various, it occurred that the intended products could not be produced in some cases by only controlling the concentration, temperature, time, etc. Thus, the modification of the enzymatic activity of MTG has become necesssary.

[0007] For modifying the enzymatic activity of MTG, it is required to produce a MTG mutant and to evaluate the activity, substrate specificity, etc. of the mutant to find out a good mutant. For producing mutants, it is necessary to manipulate wild type genes and, accordingly, it is prerequisite that a recombinant protein can be prepared. For MTG, a large-scale expression system using E. coli has been established (JP-Kokai No. 6-30771). However, MTG thus expressed in a large amount in E. coli is accumulated as insoluble protein inclusion bodies in the microbial cells. The denatured MTG in the protein inclusion body must be activated by refolding it after solubilizing it. Thus, it takes 2 to 3 weeks to prepare one mutant MTG. It has therefore been required to develop a method for reasonably modify MTG using some technique other than the techniques of randomly introducing the mutations through the entire molecule.

SUMMARY OF THE INVENTION

[0008] The object of the present invention is to provide a method for improving MTG.

[0009] Another object of the present invention is to provide a transglutaminase having an improved reactivity on substrates.

[0010] After intensive investigations made for the purpose of solving the above-described problems, the inventors have found that a transglutaminase improved in the reactivity on a substrate can be prepared on the basis of the three-dimensional structure. The present invention has been completed on the basis of this finding.

[0011] Namely, the summary of the present invention is as follows:

[0012] (1) A method for designing and preparing a mutant transglutaminase by extrapolating the binding site of MTG for a substrate on the basis of the three-dimensional structure obtained by X-ray crystal structure analysis and replacing, inserting or deleting amino acid residues positioned at the extrapolated substrate-binding site of the transglutaminase.

[0013] (2) A mutant MTG having modified substrate specificity, in which at least one of the following positions (residues positioned within 20 Å from active residue Cys 64 and exposed on the surface of the presumed binding site for acyl acceptor) in MTG amino acid sequences indicated as SEQ ID NO: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296,-297 and 300-304.

[0014] (3) A mutant transglutaminase having transglutaminase activity and modified substrate specificity, wherein when the alignment of the sequence of said mutant transglutaminase with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out, at least one of the positions corresponding to the following positions (residues positioned within 20 Å from active residue Cys 64 and exposed on the surface of the presumed binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID NO: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296,-297 and 300-304.

[0015] (4) A mutant transglutaminase having the transglutaminase activity and a modified substrate specificity, wherein when the alignment of the sequence of said mutant transglutaminase with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out by treading method, at least one of the positions corresponding to the following positions (residues within 20 A from active residue Cys 64 and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID NO: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296,-297 and 300-304.

[0016] (5) A mutant MTG or a mutant transglutaminase having changed substrate specificities through reducing the negative charge by modifying acidic amino acid residues located at the positions defined in any one of the above items (2) to (4) or amino acid residues adjacent to said acidic amino acid residues.

[0017] (6) A gene encoding the mutant MTG or the mutant transglutaminase defined in any one of the above items (2) to (5).

[0018] (7) A recombinant DNA containing the gene defined in above item (6).

[0019] (8) A microorganism harboring the recombinant DNA defined in above item (7).

[0020] (9) A method for producing a mutant MTG or a mutant transglutaminase, which comprises culturing the microorganisms defined in item (8) and collecting the mutant MTG or the mutant transglutaminase.

[0021] (10) A monoclinic MTG crystal belonging to P2₁ space group.

[0022] Namely, the present invention provides a method for rationally designing and preparing a mutant MTG on the basis of the three-dimensional structure of MTG, and the mutant MTG thus prepared.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a ribbon model showing the crystal structure of MTG.

[0024]FIG. 2 shows the surface roughness of the crystal structure of MTG observed in the same direction as that in FIG. 1.

[0025]FIG. 3 shows the alignments of amino acid sequences of MTG and TG from Streptoverticillium cinnamoneum.

[0026]FIG. 4 shows the alignments of amino acid sequences of MTG and TG from Streptoverticillium lydicus.

[0027]FIG. 5 shows the primer sets used for the site-directed mutagenesis of S2Y·S2R ·S2D mutants.

[0028]FIG. 6 shows the primer sets used for site-directed mutagenesis of del 1-2 and del 1-3 mutants.

[0029]FIG. 7 shows 1H-15N HSQC spectrum of ovalbumin reacted with the wild type in the presence of ¹⁵NH₄Cl.

[0030]FIG. 8 shows the peak intensity of signal “a” in FIG. 7 in the glutamine residues of ovalbumin reacted with the wild type, Ser type, S2R, del 1-2 or del 1-3 in the presence of ¹⁵NH₄Cl, plotted vs. the reaction time.

[0031]FIG. 9 shows the peak intensity of signal “a” in FIG. 7 in the glutamine residues of ovalbumin reacted with the wild type, S2Y, S2R or S2D mutant in the presence of ¹⁵NH₄Cl, plotted vs. the reaction time.

[0032]FIG. 10 shows ¹H-¹⁵N HSQC spectrum of ovalbumin reacted with the wild type, Sg4 or Sg7 in the presence of ¹⁵NH₄Cl.

[0033]FIG. 11 shows the atomic coordinates (1) of MTG.

[0034]FIG. 12 shows the atomic coordinates (2) of MTG.

[0035]FIG. 13 shows the atomic coordinates (3) of MTG.

[0036]FIG. 14 shows the atomic coordinates (4) of MTG.

[0037]FIG. 15 shows the atomic coordinates (5) of MTG.

[0038]FIG. 16 shows the atomic coordinates (6) of MTG.

[0039]FIG. 17 shows the atomic coordinates (7) of MTG.

[0040]FIG. 18 shows the atomic coordinates (8) of MTG.

[0041]FIG. 19 shows the atomic coordinates (9) of MTG.

[0042]FIG. 20 shows the atomic coordinates (10) of MTG.

[0043]FIG. 21 shows the atomic coordinates (11) of MTG.

[0044]FIG. 22 shows the atomic coordinates (12) of MTG.

[0045]FIG. 23 shows the atomic coordinates (13) of MTG.

[0046]FIG. 24 shows the atomic coordinates (14) of MTG.

[0047]FIG. 25 shows the atomic coordinates (15) of MTG.

[0048]FIG. 26 shows the atomic coordinates (16) of MTG.

[0049]FIG. 27 shows the atomic coordinates (17) of MTG.

[0050]FIG. 28 shows the atomic coordinates (18) of MTG.

[0051]FIG. 29 shows the atomic coordinates (19) of MTG.

[0052]FIG. 30 shows the atomic coordinates (20) of MTG.

[0053]FIG. 31 shows the atomic coordinates (21) of MTG.

[0054]FIG. 32 shows the atomic coordinates (22) of MTG.

[0055]FIG. 33 shows the atomic coordinates (23) of MTG.

[0056]FIG. 34 shows the atomic coordinates (24) of MTG.

[0057]FIG. 35 shows the atomic coordinates (25) of MTG.

[0058]FIG. 36 shows the atomic coordinates (26) of MTG.

[0059]FIG. 37 shows the atomic coordinates (27) of MTG.

[0060]FIG. 38 shows the atomic coordinates (28) of MTG.

[0061]FIG. 39 shows the atomic coordinates (29) of MTG.

[0062]FIG. 40 shows the atomic coordinates (30) of MTG.

[0063]FIG. 41 shows the atomic coordinates (31) of MTG.

[0064]FIG. 42 shows the atomic coordinates (32) of MTG.

[0065]FIG. 43 shows the atomic coordinates (33) of MTG.

[0066]FIG. 44 shows the atomic coordinates (34) of MTG.

[0067]FIG. 45 shows the atomic coordinates (35) of MTG.

[0068]FIG. 46 shows the atomic coordinates (36) of MTG.

[0069]FIG. 47 shows the atomic coordinates (37) of MTG.

[0070]FIG. 48 shows the atomic coordinates (38) of MTG.

[0071]FIG. 49 shows the atomic coordinates (39) of MTG.

[0072]FIG. 50 shows the atomic coordinates (40) of MTG.

[0073]FIG. 51 shows the atomic coordinates (41) of MTG.

[0074]FIG. 52 shows the atomic coordinates (42) of MTG.

[0075]FIG. 53 shows the atomic coordinates (43) of MTG.

[0076]FIG. 54 shows the atomic coordinates (44) of MTG.

[0077]FIG. 55 shows the atomic coordinates (45) of MTG.

[0078]FIG. 56 shows the atomic coordinates (46) of MTG.

[0079]FIG. 57 shows the atomic coordinates (47) of MTG.

[0080]FIG. 58 shows the atomic coordinates (48) of MTG.

[0081]FIG. 59 shows the atomic coordinates (49) of MTG.

[0082]FIG. 60 shows the atomic coordinates (50) of MTG.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0083] The present invention will be concretely described thereinafter.

[0084] Transglutaminase is widely used for the production of foods such as gelatins, cheeses, yogurts, tofu, boiled fish pastes, hams, sausages and noodles and also for improving the quality of meats (JP-Kokai No. 64-27471). Transglutaminase is also used for various industrial purposes, for example, in the production of materials for heat stable microcapsules and the production of carriers for immobilized enzymes. Transglutaminase catalyzes the acyl transfer reaction of γ-carboxamide group of glutamine residue present in the peptide chain of a protein molecule. When ε-amino group of lysine residue in a protein molecule acts as an acyl acceptor, ε-(γ-Glu)-Lys bond is formed in the protein molecule and between the molecules.

[0085] The inventors found that transglutaminase does not react with all glutamine residues, namely, the possibility of the reaction, i.e. reactivity of transglutaminase varies depending on the kinds and positions of the residues surrounding the glutamine residues. Therefore, modification of the reactivity of transglutaminase on the glutamine residues under specified environment lead to the modification of the gelation rate and, as a result, the range of the use of transglutaminase may be further widened.

[0086] In the present invention, for modifying the substrate specificity of MTG, the three-dimensional structure thereof is determined by X-ray crystal structure analysis and the manner of binding to the substrate is predicted and the mutations suitable for the purpose are designed and introduced. The term “substrate specificity” herein used indicates the reactivity with each glutamine residue in the acyl acceptor.

[0087] The determination of the three-dimensional structure of a protein by X-ray crystal structure analysis will be carried out by the following procedure:

[0088] (1) The protein is crystallized. The crystallization is indispensable for determining the three-dimensional structure. In addition, the crystallization is industrially useful for the purification of a protein in high purity and also for a method of stable storage of a protein in high density, which method is highly resistant against proteases.

[0089] (2) The crystals thus obtained are irradiated with X-ray to collect the diffraction data. In many cases, protein crystals are damaged by the X-ray irradiation which reduces the diffraction capacity thereof. In such cases, low temperature measurement technique has recently been spread wherein the crystals are rapidly cooled to about −173° C. and the diffraction data are collected in this state. Finally, the synchrotron radiation with high intensity is utilized for collecting the high-resolution data used in the determination of the structure.

[0090] (3) For the analysis of the crystal structure, the phase information is required in addition to the diffraction data. The MTG structure cannot be determined by molecular replacement method because the crystal structures of analogous proteins are yet unknown. Thus, the phase problem must be solved by heavy atom isomorphous replacement method. This method comprises introducing a metal atom having a large atomic number such as mercury or platinum into the crystal and obtaining the phase information taking advantage of the contribution of the large X-ray scattering capacity of the metal atom to the X-ray diffraction data. The phase thus determined can be improved by smoothing the electron density in the solvent region in the crystal. Since the electron density of water molecules in the solvent region is scarcely observed because of their have high fluctuation, the electron density very close to the true electron density can be obtained by approximating the electron density in this region to 0, which improves the phase. When plural molecules are contained in an asymmetric unit, the phase will be further remarkably improved by averaging the electron density of these molecules. A protein model is fit to the electron density map calculated using thus improved phase. This process is carried out using a program such as QUANTA (MSI Co., U.S.A.) by computer graphics. Then the structure is refined using a program such as X-PLOR (MSI Co., U.S.A.) to complete the structure analysis.

[0091] The inventors crystallized purified natural MTG from Streptoverticillium mobaraense and recombinant MTG (Met type) with additional Met originating from initiation codon at the N-terminal, but could not obtained a crystal suitable to be analyzed. However, because better crystals were obtained with natural MTG rather than with Met-type MTG, the inventors prepared Asp-type MTG (Example 1) after having considered that the recombinant MTG (Asp type MTG) starting with Asp1, like the natural one, obtained by removing Met at the N-terminal, might have the possibility of resulting in the crystals tolerable to the analysis. As a result, Asp-type MTG yielded a good crystal (Example 2), and the three-dimensional structure was successfully obtained (Example 3). The atomic coordinates in the MTG three-dimensional structure are shown in FIGS. 11 to 60.

[0092] The reaction mechanism of transglutaminase is considered to be as follows: An acyl donor containing Gln reacts with a transglutaminase to form a reaction intermediate. Then an acyl acceptor containing Lys attacks the intermediate to form ε-(γ-Glu)-Lys bond between the acyl donor and the acyl acceptor. For using a protein as the acyl donor, it is not sufficient that it contains Gin, but it is required that the region around Gln can be bound appropriately to the substrate-binding site of MTG. On the contrary, it is enough in most cases for the protein to contain a primary amine to serve as the acyl acceptor. Therefore, modifying the substrate specificity of MTG means modifying the structure of the binding sites for the acyl donor. If MTG can be modified so as to reduce the substrate specificity thereof or, in other words, so as to accept a larger amount of the acyl donor, the cases increases wherein the joining of proteins may be possible using a smaller amount of MTG, and therefore, the reduction of the amount used is expected. Further, if the joining of proteins in which the cross-linking is presently impossible become possible, the application of MTG in a new field will become possible. The three-dimensional structure of MTG is indispensable for extrapolating the binding manner between MTG and the acyl donor.

[0093] MTG molecule elucidated in the present invention is in the shape of discoid form having a size of 65×59×41 Å (see FIGS. 1 and 2). Namely, in FIG. 1, the crystal structure of MTG is shown by a ribbon model. α-helices and β-sheets are shown by spirals and arrows, respectively. An active residue, Cys⁶⁴, is shown by boll and stick model (upper central part).

[0094]FIG. 2 shows the surface concavoconvex of the crystal structure of MTG in the same direction as in FIG. 1. The regions having the electric charge are indicated in dark color and those free of the electric charge are indicated in light color. Observing the disk from upper side, it has a wedge-shaped cleft. In the inner part of the cleft, active residue Cys⁶⁴ is located. The fact that Asp²⁵⁵ is positioned in the neighborhood of the active residue Cys⁶⁴ suggests that SH group of Cys⁶⁴, which may be dehydrogenated by Asp²⁵⁵, causes the reaction. The side chain of Cys⁶⁴ faces the solution side and it can approach the acyl donor and acyl acceptor. In Ca²⁺-dependent human blood coagulation factor XIII, the active residue Cys is not exposed to the solvent. Therefore, for binding with the acyl donor, human blood coagulation factor XIII must undergo a significant structure change for the active residue to bind to the acyl donor. On the other hand, MTG is considered to be able to bind to the acyl donor without changing its structure. Therefore, it is supposed that MTG can use more Gln residues as the substrates than human blood coagulation factor XIII uses. Further, MTG has a merit in that a mutant having further reduced substrate specificity can be easily designed, because it is not required to take the structural change associated with the binding to the acyl donor into consideration. Furthermore, the molecular weight of MTG is as low as 38,000, while that of human blood coagulation factor XIII is 166,000, which make the determination of the binding site of MTG for the acyl donor easy. By observing the structure of MTG, it would be understood that the substrate is very likely to bind in the wedge-shaped cleft leading from the solvent to Cys⁶⁴.

[0095] Many hydrophobic amino acid residues and aromatic amino acid residues are positioned in the cleft leading to active residue Cys⁶⁴, and thus the mutual action thereof with hydrophobic residues or aromatic residues in the acyl donor is considered to be one of the keys for the determination of the substrate specificity. The polar resides including Asp are also scattered on the cleft surface, which suggests that they play an important role in the recognition of the acyl donor. A mutant transglutaminase having a modified substrate specificity can be obtained by the replacement, insertion or deletion of amino acid residues located in the cleft which is supposed to be the acyl donor binding region, preferably by the replacement of the polar residues (Asp, Glu, Lys and Arg) or aromatic residues (Phe, Tyr and Trp) with other amino acids, the replacement thereof with polar residues or aromatic residues, the deletion of the polar residues or aromatic residues or insertion of polar residues or aromatic residues. It is to be noted, however, since it is supposed that the activity will be lost by modifying Cys⁶⁴ and Asp²⁵⁵ which play an important role for the activity, these residues should be excluded in the production of the mutants.

[0096] As shown in Examples given below, acidic amino acids present adjacent to the reactive site in the substrate proteins. Glutamine residues reactive with MTG are positioned on the surface of the substrate protein in many cases and it is considered that acidic amino acids which may easily exposed to the solvent are likely to present around them. When acidic residues are present at the interaction site of the substrate protein of MTG, the negative charge in MTG and that in the substrate protein repel each other to reduce the enzymatic reaction velocity. The negative charge can be removed by modifying the acidic amino acid residues exposed on the surface of the cleft of MTG containing Cys⁶⁴ which is supposed to be bound by the acyl donor or, alternatively, the negative charge can be relaxed by converting amino acid residues sequentially or spatially adjacent to the acidic amino acids into basic amino acids to obtain the transglutaminase having a modified substrate specificity. Such acidic amino acids include the residues Nos. 1, 3, 4, 28, 58, 249, 300 and 304 of MTG described-in SEQ ID NO: 2.

[0097] The amino acids adjacent to the acidic amino acids are the amino acid residues within 5 Å from carboxyl groups of the acidic amino acids such as aspartic acid and glutamic acid, and are preferably amino acid residues having the degree of exposition to the solvent of at least 10%. They include residues Nos. 2, 5, 59, 248-250, 252, 278, 283-285, 289, 291, 296-297 and 302-303 of MTG described in SEQ ID NO: 2.

[0098] The amino acid sequence of MTG is shown in SEQ ID NO: 2, and the nucleotide sequence of the gene encoding MTG is shown in SEQ ID NO. 1.

[0099] As shown in Example 5, it was confirmed that the reaction rate on a particular glutamine residue in a protein molecule could be elevated by the deletion of Asp¹ or Asp³ or by the replacement of Ser2. Because the distance from active residue Cys⁶⁴ to Asp¹ at the N-terminal is 20 Å, it is suggested that there is a possibility that the substrate specificity can be modified by modifying the amino acid residues located within 20 Å from Cys⁶⁴. In addition, because it was confirmed that at least the loop regions (233-253 and 276-288) defined the substrate specificity, it was considered that the substrate specificity could be modified by modifying these regions. The possibility of changing the substrate specificity is further increased by modifying the residues exposed on the surface of the cleft having Cys⁶⁴ supposed to be bound by the acyl donor. To know whether the amino acid residues are exposed to the solvent or not, the solvent accessibility may be calculated using a program such as QUANTA. The solvent accessibility is the value resulting from dividing the exposure area to the solvent of a residue in the protein by the exposure area to the solvent of the amino acid in its free form, which is indicated as a percentage. The possibility of changing the substrate specificity of MTG may be high when the residues existing within 20 Å from Cys⁶⁴ and having a solvent accessibility of at least 10% are modified. Such residues correspond to residue Nos. 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296-297 and 300-304 of MTG described in SEQ ID NO. 2.

[0100] In designing and producing a mutant transglutaminase, the codon in the gene encoding the transglutaminase corresponding to the amino acid residues at the above-described substrate-binding site may be modified by site-directed mutagenesis and the obtained gene may be introduced into a suitable vector and transferred into a host, and then the transformant may be cultured. The intended mutant transglutaminase may be recovered from the transformed cultured microbial cells of and evaluated.

[0101] The site-directed mutagenesis methods for introducing intended mutation at the intended sites of DNA include, for example, a PCR method shown in Examples [Higuchi, R., 61,in PCR technology; Stockton Press edited by Erlich, H. A. (1989); Carter P., Meth. In Enzymol., Vol. 154, p. 3382 (1987)]; and a method wherein a phage is used [Kramer, W. and Frits, H. J., Methods in Enzymology, Vol. 54, p. 350 (1987), Kunkel, T. A. et al., Methods in Enzymology, Vol. 154, p. 367 (1987)]. The vectors for introducing the genes encoding mutant transglutaminase are not particularly limited so far as they can be replicated in hosts. When Escherichia coli is used as a host, plasmids capable of autonomously replicating in this bacterium can be used. For example, pUC19, pET or pGEMEX can be used.

[0102] Preferred hosts are, for example, strains of Escherichia coli, however, any bacterium can be used as the host so far as the replication origin of the constructed recombinant DNA and the mutant transglutaminase genes can function, the mutant DNA can be replicated and the expression of the mutant transglutaminase genes can be expressed in the bacterium. The representative preferred host is Escherichia coli BL21(DE3)pLysS when T7 promoter is used or Escherichia coli JM109 in other cases.

[0103] An ordinary method can be employed, without any particular limitation, for introducing the recombinant DNA obtained by ligating the vector with the DNA fragment containing the gene encoding the mutant transglutaminase into the host. When Escherichia coli is used as the host, there can be employed calcium chloride method [J. Mol. Biol., Vol. 53, p. 159 (1970)], Hanahan method [J. Mol. Biol., Vol. 166, p. 557 (1983)], SEM method [Gene, Vol. 96, p. 23 (1990)] and Method of Chung et al. [Proceedings of the National Academy of Sciences of the USA, Vol. 86, p. 2172 (1989)].

[0104] MTG may be expressed in the bacterial cell at a high level by culturing the transformant harboring the recombinant DNA containing the gene encoding the mutant transglutaminase in a suitable medium containing a carbon source, a nitrogen source, an inorganic ion and, if necessary, further containing an organic nutrient source. For extracting the mutant transglutaminase from the cultured cells, the cells are collected after culturing them, suspended in a buffer solution, and subjected to the lysozyme treatment, freezing-and-thawing treatment, ultrasonic disruption, etc., and then, they are disrupted and centrifuged to separate the suspension into the supernatant and the precipitant.

[0105] Since the mutant transglutaminase is produced in the form of a protein inclusion body and separated as the precipitant, it may be solubilized with a denaturing agent or the like and then the denaturing agent may be removed to isolate and purify the protein. The denaturing agents for solubilizing the produced protein inclusion body may be, for example, urea (for example, 8M) and guanidine hydrochloride (for example, 6 M). By reducing the concentration of the denaturing agent by dilution or the like, the protein having transglutaminase activity is regenerated. The solutions used for the dilution may include phosphate buffers and Tris buffers.

[0106] After regenerating the activity, the active protein can be isolated and purified by a suitable combination of known isolation method and purification method. Examples of these methods are salting out, dialysis, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase high-performance chromatography.

[0107] The term “substrate specificity” in the context of the present invention indicates the reactivity of each glutamine residue in the substrate protein molecule to a transglutaminase. The change in the substrate specificity of a transglutaminase means the change in the reactivity of at least one glutamine residue. When the mutant transglutaminase having the changed substrate specificity is reacted on a protein, the degree of crosslinking of the protein, namely, the degree of the gelation, is also changed compared with the results obtained by reacting the original transglutaminase.

[0108] Methods for comparing the substrate specificities of transglutaminases include, for example, the method wherein the gelation time of casein is determined, the method wherein the cross-linked polymer obtained after the transglutaminase reaction is analyzed by electrophoresis, and the method wherein the reaction product is fragmented and then analyzed. It is also possible that the substrate protein is reacted with ¹⁵N-labeled ammonium ion by using the transglutaminase and then the labeled glutamine residues are detected by NMR (JP-Application No. 2000-141152).

[0109] The analysis of substrate specificity by the latter method is the method of comparing the reactivity of individual glutamine residue in the protein, taking advantage of the fact that the carboxamide nitrogen in the glutamine residue, which functions as a substrate, is ¹⁵N-labeled by a transglutaminase. The substrate specificities of the transglutaminases can be compared with each other, taking advantage of that glutamine residue which acts as a substrate for transglutaminase is specifically ¹⁵N-labeled and that the labeling rate varies depending on the reactivity of the transglutaminase to the glutamine residue.

[0110] The unit activity of transglutaminase is determined and defined as follows: The reaction is carried out using benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as the substrates, the resulting hydroxamic acid is converted into an iron complex hereof in the presence of trichloroacetic acid and then the amount thereof is determined as an absorbance at 525 nm. Thus the calibration curve is prepared based on the amount of hydroxamic acid. The amount of the enzyme which produces 1 μmole of hydroxamate per one minute is defined to be the unit of transglutaminase activity, one unit. The details of this determination method were previously reported (for example, JP-Kokai No. 64-27471).

[0111] Besides the transglutaminase from Streptoverticillium mobaraence (MTG), it is also possible to modify an enzyme which has the transglutaminase activity and amino acid sequence homologous to MTG, or an enzyme which has the transglutaminase activity and which is supposed to have the three-dimensional structure similar to that of MTG, on the basis of the three-dimensional structure of MTG. The amino acid replacement effective for the modification of the substrate specificity or the like of MTG is also effective for analogous enzymes such as those from Streptoverticillium cinnamoneum and Streptoverticillium lydicus (JP-Kokai No. 10-504721). The amino acid sequence of the transglutaminase from Streptoverticillium cinnamoneum is shown in SEQ ID NO: 6, the nucleotide sequence of the gene encoding the amino acid sequence is shown in SEQ ID NO: 5, the amino acid sequence of the transglutaminase from Streptomyces lydicus is shown in SEQ ID NO: 8, and the nucleotide sequence of the gene encoding the amino acid sequence is shown in SEQ ID NO: 7. In this connection, the homology of the amino acid sequence between MTG and transglutaminase from Streptoverticillium cinnamoneum is 78% (FIG. 3), and that of the amino acid sequence between MTG and transglutaminase from Streptomyces lydicus is 79% (FIG. 4). Namely, FIG. 3 shows the alignment of amino acid sequences of MTG and transglutaminase from Streptoverticillium cinnamoneum. Conserved amino acid residues are shown by “*”. FIG. 4 shows the alignment of amino acid sequences of MTG and the transglutaminase from Streptoverticillium lydicus. Conserved amino acid residues are shown by “*”.

[0112] The coordinate assignment of amino acid residues of two different proteins may be revealed from the alignment of the amino acid sequences (Sequence Alignment) when the homology between the amino acid sequences of them is above about 20%, and it may be revealed from the three-dimensional structures and the alignment of the amino acid sequences (Threading) when the homology is below about 20%. The former can be carried out using a program such as BLAST and the latter can be carried out using a program such as INSIGHT II. BLAST can be obtained by using ftp as the file adapted to the computer to be used among the files existing in “/blast/executable” of ncbi.nlm.nih.gov. The operating instructions are described in detail in http:///genome.nhgri.nih.gov/blastall/blast install. INSIGHT II is commercially available (MSI Co.).

[0113] A mutant transglutaminase having modified substrate specificity is usable for the preparation of gelled foods such as jellies, as well as yogurts, cheeses and gelled cosmetics; for the improvement in the quality of meats; for the production of materials of thermally stable microcapsules and carriers for immobilized enzymes; and for the enzymatic labeling of proteins. When a protein is ¹⁵N-labeled, the structure analysis by NMR is made possible (JP-Application No. 2000-141151), and when it is labeled with a primary amine, the solubility and stability thereof can be improved (JP-Application No. 2000-141152). Proteins usable as the substrates for the transglutaminase may vary. Thy include those constituting muscle such as actin and myosin; human plasma components such as albumin, immunoglobulin and blood coagulation factors; enzymes such as protease and transferase; hormones such as growth hormone and erythropoietin; cell growth factors for proliferating or inhibiting cells; immune reaction modulators for cell differentiation, induction and stimulation; and biologically active proteins produced by cells, such as monokine, cytokine and lymphokine. The origins of those proteins are not particularly limited and the proteins may be derived from animals, vegetables and microorganisms. Further, the proteins may be those expressed in Escherichia coli, yeast and animal cells by introducing the genes of these proteins into them or those expressed by using a cell-free protein synthesis system.

[0114] The reaction of such a protein in the presence of the transglutaminase is carried out under conditions where the transglutaminase can function. For example, the substrate protein and the transglutaminase are kept at a pH in the range of about 5.0 to 9.0, preferably abut 6.0 to 8.0 and at a temperature in the range of about 4 to 55° C., preferably about 25 to 40° C. in an aqueous solvent. In this reaction, the concentration of the substrate protein is preferably in the range of 1 μM to 1 M, and the amount of the transglutaminase is desirably in the range of about 10 nM to 100 μM which corresponds to about 0.01 to 20 units per mmol of the protein. However, the reaction conditions are not limited to those described above.

[0115] The range of the industrial utilization of MTG can be further widened when the heat stability and optimum pH can be modified in addition to the modification of the substrate specificity. If the heat stability is improved, the gelation of protein at a higher temperature may become possible. In the production of tofu (bean curd) filled into a pack, which is commonly sold in supermarkets, MTG is added to soybean milk and then the obtained mixture is tightly closed in the pack and then heated. Therefore, the temperature easily elevates to a point at which MTG is denatured. If the heat stability of MTG can be improved, the amount of protein inactivated by denature can be reduced, whereby the cost is lowered. The thermal stability can be improved by, for example, the introduction of proline residue, the replacement of the residues forming the counterclockwise helix structure with glycine residues [Protein Engng, Vol. 6, pp. 85-91 (1993)], or filling of the cavities in the protein [Biochemistry, Vol. 32, pp. 6171 to 6178 (1993)]. Further, if the optimum pH can be shifted to the alkaline region, the gelation efficiency of a substance which reacts under an alkaline reaction condition can be elevated such as the case of udon which are prepared by joining gluten. The modification of optimum pH can be achieved by modifying pKa of the active residues [Protein Engineering, Vol. 11, pp. 383-388 (1998)].

EXAMPLES

[0116] The following Examples will further illustrate the present invention, which by no means limit the invention.

Example 1

[0117] Preparation of Recombinant Native Type (Asp Type) MTG

[0118] MTG expression plasmid pETMTGXa-01 which will be described below was constructed to produce recombinant MTG having the same N-terminal amino acid sequence as that of native type in Escherichia coli. pETMTGXa-01 is a plasmid capable of expressing a fused protein having the recognition sequence (IEGR) of factor Xa between the amino terminal region of T7 gene 10 protein and MTG. The procedures for constructing pETMTGXa-01 are described hereinafter. Firstly, primer pGEXMTGF01 (SEQ ID NO: 9 in the sequence listing) containing the sequence encoding BamHI cleavage site and Factor Xa recognition site at the upstream side of MTG gene and also primer pGEXMTGR01 containing Sac I cleavage site (SEQ ID NO: 10 in the sequence listing) were synthesized. PCR was carried out using these primers and MTG genes in MTG expression plasmid pUCTRPMTG-02(+) (JP Kokai No. 11-75876) as a template, and the amplified fragment was cloned in PGEM-T Easy Vector (Promega). PCR were carried out by 25 cycles of “96° C. for 30 seconds, 50° C. for 15 seconds and 60° C. for 1 minute”. The cloning of the amplified fragment was carried out according to the instructions of supplier. Clone in which the amplified fragments were inserted in an opposite direction to lacZ gene were selected. The nucleotide sequences of the plasmids in the clones were analyzed and the plasmid having the intended correct sequence was named “pGEMMTGXa”. Then pET5a (Takara Shuzo Co., Ltd.) vector was cleaved by EcoRI, bunt-ended, and cleaved by BamHI. pGEMMTGXa was cleaved by SalI, blunt-ended, cleaved by BamHI, and the obtained fragment containing MTG gene was subcloned in the above-described enzymatically digested pET5a to construct pETMTGXa-01.

[0119]Escherichia coli BL21(DE3)pLysS (Promega) having pETMTGXa-01 introduced therein was inoculated in 50 ml of L medium (10 g/L of Tryptone, 5 g/L of yeast extract, 5 g/L of NaCl and 1 g/L of glucose; pH: 7.2) containing 200 μg/ml of ampicillin and pre-cultured at 37° C. for 6 hours. Then, 8 ml of the pre-culture was subcultured for 4 hours at 37° C. in M9 casamino acid medium (8 g/L of casamino acids, 5 g/L of ammonium chloride, 0.2 g/L of yeast extract, 2 mg/L of vitamin B₁ hydrochloride, 14.5 mg/L of calcium chloride dihydrate, 15.1 g/L of disodium hydrogen phosphate 12 hydrate, 3 g/L of potassium dihydrogen phosphate, 0.5 g/L of magnesium sulfate heptahydrate and 5 g/L of glucose; wherein magnesium sulfate heptahydrate and glucose were separately sterilized) containing 200 μg/ml of ampicillin. When OD₆₆₀ reached to about 2.0, 0.8 ml of 1M IPTG was added to the culture, and the culture was further continued at 37° C. for 14 hours.

[0120] The cells were collected from the broth by centrifugation and suspended in 200 ml of a mixture of 20 mM Tris-HCl, 30 mM of NaCl and 5 mM of EDTA, pH 7.5. 1 mg/ml of lysozyme was added to the obtained suspension. After leaving the resultant mixture at 4° C. for 1 hour, the cells were crushed by ultrasonication. Protein inclusion bodies containing denatured MTG were collected by centrifugation. The protein inclusion bodies were thoroughly suspended in a small amount of water. Reagents and water were added to the obtained suspension so that the suspension contains 8 M of urea, 20 mM of sodium phosphate, 1 mM of EDTA and 20 mM of DTT/30 ml to dissolve the protein inclusion bodies. After incubating at 37° C. for 2 hours, pH was lowered to 4 by adding dropwise 1 M of hydrochloric acid, and the precipitant was removed after centrifugation. The concentration of the solubilized MTG was determined, which was found to be about 20 mg/ml. 20 ml of the above solubilized MTG solution were slowly added dropwise to 1000 ml of 20 mM sodium acetate, pH 4.0, containing 2 mM DTT, and the mixture was left to stand for 2 hours. After 2 hours, 4 M of sodium hydroxide was added dropwise to the mixture to elevate pH to 6 to complete the refolding. The concentration of MTG was determined to be about 0.14 mg/ml. The refolding procedure was totally carried out in a cold room at 4° C.

[0121] The total solution thus obtained was concentrated to a volume of 200 ml by ultrafiltration (Hydrosart, 10 kDa Molecular cut off; Sartocon Slice Casette, Sartorius). and the solvent was replaced with 20 mM Tris-HCl (pH 7.5) by using Sephadex G25 (M) (Pharmacia Aktiebolag). 5 mg of bovine factor Xa (Hematologic Technology Inc.) was added to 250 ml of the obtained solution and then the mixture was left to stand overnight at 5° C. The concentration of MTG was determined to be about 0.36 mg/ml. The N-terminal amino acid sequence of MTG cleaved by factor Xa was analyzed with a protein sequencer (Shimadzu Corporation) to confirm that the sequence attached to the N-terminal had been completely cleaved by factor Xa and also that there was only the recombinant MTG having the mature type N-terminal amino acid sequence.

[0122] After the cleavage with factor Xa, pH of the solution was adjusted to 5.8 with 1 M hydrochloric acid and then the solution was diluted 5-fold with a buffer solution (20 mM sodium acetate, pH 5.8) used for equilibration of cation exchange chromatography. The obtained solution was applied to the cation column (CM Sepharose FF, 2.6φ10 cm; Amersham Pharmacia Biotech) sufficiently equilibrated with the buffer solution. After the re-equilibration with the same buffer solution, a protein fraction (46 ml) eluted according to the linear concentration gradient of 0-0.4 M NaCl was recovered using UV absorption at a wavelength of 280 nm as an index. The recovered fraction was diluted 10-fold with a buffer solution (20 mM sodium acetate, pH 5.5) used for the equilibration of the cation exchange chromatography. ⅓ of the diluted solution was applied to the cation column (6 ml of Resource S; Amersham Pharmacia Biotech) sufficiently equilibrated with the same buffer solution. After the re-equilibration with the same buffer solution, a protein fraction eluted according to the linear concentration gradient of 0-0.5 M NaCl was fractionated using UV absorption at a wavelength of 280 nm as an index. A fraction (2 ml) in which no impurities were detected by reversed-phase HPLC and which was free from low pI form of MTG was recovered from the front side of the peak top. The similar purification was carried out twice, and the side fraction of the recovered fraction was re-chromatographed to finally recover the fraction of 8 ml (40 mg). Again, the solvent was replaced with 20 mM phosphoric acid, pH 6.0 by using Sephadex G25(M). The recovered fraction was analyzed by isoelectric focusing to confirm that it was the recombinant MTG of a high purity substantially free from analogues having different pl. All of the chromatography procedures were carried out at room temperature.

Example 2

[0123] Crystallization

[0124] The crystallization was carried out using the hanging drop mode of vapor diffusion method. Recombinant Asp type MTG solution (concentration: 15 mg/ml) and 83 mM buffer (pH 5.0) of sodium cacodylate containing 25% polyethylene glycol 1000 and 25 mM calcium chloride were dropped in the same amounts (2 μl) on a siliconized cover glass and mixed together. The cover glass was placed on the buffer in such a manner that the drops of the mixed liquid were hanging over the well filled with 500 μl of the buffer, and they were left to stand at 20° C. Crystals were formed one week after. 10 days after, the crystals grew into plate-like crystals of a measurable size (about 0.5×0.3×0.1 mm). The MTG crystal was analyzed at a low temperature, because MTG crystal would be damaged by X-ray which leads to the gradual reduction of resolution when analyzed at ambient temperature. The crystal was transferred into 117 mM buffer (pH 5.0) of sodium cacodylate containing 35% of polyethylene glycol 1000 and 35 mM of calcium chloride and rapidly cooled by blowing nitrogen gas at −173° C.

[0125] X-ray diffraction data of 2.7 Å resolution were obtained with a X ray diffractometer, R-AXIS IIc (Rigaku Co., Ltd.) and the crystallographic parameters were determined. The space group was P2₁, lattice constants were as follows: a=78.4 Å, b=117.1 Å, c=85.7 Å and β=112.90. Assuming that the asymmetric unit contains 4 molecules (molecular weight: 38,000), water content of the crystals was calculated at 50%.

Example 3

[0126] Determination of Three-Dimensional Structure

[0127] The crystals were immersed in a solution of heavy metal salts and the heavy atom derivatives were screened. The diffraction data of the heavy atom derivative crystals were obtained by using SMART6000 (Bruker) and the synchrotron radiation facilities BL-6B of the National Laboratory for High Energy Physics. From difference Patterson map compared with the native data, it was shown that EMTS, K₂OSCl₆ and K₂IrCl₆ provided good heavy atom isomorphous-replacement crystals. Major heavy atom sites were determined from the differential Patterson maps of the respective heavy atom isomorphous replacement crystals, and then, other minor heavy atom sites were determined using the difference Fourier maps. The coordinates of these heavy atom sites were refined by the phase determination program “MLPHARE” and the phase was calculated. The phase was improved by solvent flattering and histogram matching using the program DM and then an electron density map was prepared. In the electron density map, 4 MTG molecules were refined in an asymmetric unit. The electron density corresponding to the 4 molecules was averaged using the program DM to remarkably improve the quality of the electron density. The electron density map prepared with 2.7 Å resolution was very good and all the amino acid residues from N terminal to C terminal could be fitted into the electron density.

[0128] The first model was constructed on computer graphics using QUANTA program, and the structure was refined using X-PLOR program. The final models (FIGS. 1 and 2) refined with X-ray diffraction data of a 2.4 Å resolution at the synchrotron radiation facilities BL-6B (National Laboratory for High Energy Physics, Tsukuba,) contained all of the 331 amino acid residues and 400 water molecules. The crystallographic reliability factor (R factor) determined by using the reflection at 10 to 2.4 Å resolution was 19.6%. In a Ramachandran plot prepared with the program PROCHECK, it was shown that 82% of residues other than glycine located in the most desirable region and 15% thereof located in the second most desirable region.

Example 4

[0129] Preparation of Site-Directed Mutant

[0130] Because N-terminal Asp¹ is positioned at the entrance of the cleft, it is supposed that it has some responsibility on the recognition of the substrate (FIGS. 1 and 2). The side chain of Asp¹ has no interaction with other residues constituting the enzyme and, therefore, it is considered that even when the residue is modified, there is no risk of distorting the three-dimensional structure of the protein. The effects of Asp¹ on the substrate specificity can be examined by preparing a mutant in which this residue has been removed (hereinafter referred to as “Ser type mutant” because it starts with Ser²) and then comparing the substrate specificity thereof with that of the wild type. Because Escherichia coli obtained by transforming plasmid containing a gene encoding Ser-type mutant had been already constructed (JP-Kokai No. 11-075876), we designed to prepare the Ser-type mutant using this plasmid.

[0131] It was also supposed that the substrate specificity could be modified also by replacing Ser² located at the entrance of the channel with another amino acid. we designed to prepare three mutants, i. e. Ser2Y obtained by replacing Ser² with Tyr for the purpose of enhancing the hydrophobic mutual action and S2R and S2D obtained by replacing Ser² with Arg and Asp, respectively, for the purpose of enhancing the electrostatic interaction. These mutations were introduced into MTG having additional Met corresponding to the initiation codon at the N-terminal (Met type).

[0132] Additionally, insulin A chain (Sigma) wherein cysteine residue had been oxidized was bought as the substrate protein, and the glutamine residues which could be the substrates of MTG were examined. Insulin A chain is a protein (SEQ ID NO: 23) consisting of 21 amino acid residues, which has glutamine residues at the 5^(th) position and the 15^(th) position from the N-terminal. The reasons why insulin A chain was selected for examining the structural characteristics of the substrate protein are that it contains plural glutamine residues and the positions of the glutamine residues reacted with MTG can be easily determined because it has a low molecular weight.

[0133] 200 μl of 1 M ¹⁵NH₄Cl and 2 μl of 1 mM MTG were added to 1 mg of insulin A chain wherein the cysteine residues were oxidized and then they were stirred and subjected to mass spectrometry. The mass spectrometry was carried out during fragmenting individual amino acid residues to identify the ¹⁵N-labeled residue which was proved to be the 15^(th) glutamine residue from the N-terminal. As a result, it was found that in the two glutamine residues in insulin A chain, the 15^(th) glutamine residue was the substrate of MTG.

[0134] Glutamic acid is present at position 17 in insulin A chain. This glutamic acid residue is located adjacent to the 15^(th) glutamine residue with the insertion of one residue. Since amino acid side chains on a polypeptide chain incline to face the same direction when they are located with the insertion of one residue, the glutamine residue at position 15 and the glutamic acid at position 17 are therefore considered to be not only close to each other in the sequence but also to be spatially close to each other. Because acidic amino acid is present near the 15^(th) glutamine residue, it is not preferred that an amino acid having a negative electric charge is present at the substrate binding site of MTG. In most cases, the glutamine residues reactive with MTG are positioned on the surface of the substrate protein. It is considered that acidic amino acids which may easily exposed to the solvent may present surrounding the glutamine residue. This is also applicable to the substrate proteins other than insulin.

[0135] Accordingly, it is supposed that the substrate specificity can be improved when the negative charge is reduced by modifying the acidic amino acids or by converting the surrounding amino acids into basic amino acids to obtain a mutant transglutaminase having a modified substrate specificity. Ser-type MTG was the one in which the aspartic acid in SEQ ID No: 2 had been removed, and S2R mutant was the one in which basic amino acids were arranged near the acidic amino acids to reduce the negative charge, all of which meet the above criterion. They satisfy the above-described conditions. Additionally, del 1-2 was prepared by removing residue Nos. 1 and 2 and del 1-3 was prepared by removing residue Nos. 1 to 3 to remove the negative charge of Asp¹ and Asp³, respectively.

[0136] Because the two loops (233-253 and 276-288) which locate to surround the active residue Cys⁶⁴ are positioned as if the substrate is sandwiched by them, they are likely to define the substrate specificity. However, the loops cannot be blindly removed because they also play an important role in the folding of MTG. Therefore, the sites satisfying the following conditions (1) and (2) were searched based on the three-dimensional structure of MTG: (1) the site hardly interact with the sites other than the loops and it does not affect the folding and (2) the distance between N-terminal and C-terminal of the deleted site (residue) is not longer than 7 Å and it can be replaced with 3 glycine residues. The 3 glycine residues are mentioned only considering that the turn structure can be formed with them, and they are not restrictively mentioned. For example, because residues Nos. 241 to 252 have substantially no interaction with other sites and since the distance between N-terminal of residue No. 241 and C-terminal of residue No. 252 is about 6 Å, residue Nos. 241-252 can be replaced by 3 glycine residues. It was attempted to produce Sg4 by replacing residue Nos. 241 to 252 with 3 glycine residues and Sg7 by replacing residue Nos. 278 to 287 with 3 glycine residues as the mutants satisfying such conditions in which the loops were removed to elucidate the contribution of the two loops surrounding Cys⁶⁴ active residue.

[0137] (1) Preparation of Ser-Type MTG

[0138]Escherichia coli JM109 harboring plasmid pUCTRPMTG-02(+) containing Ser-type MTG gene (JP-Kokai No. 11-075876) having Ser-type MTG gene was inoculated into 50 ml of L medium containing 200 μl/ml of ampicillin, and pre-cultured at 30° C. for 7 hours. 16 ml of the pre-culture was transferred into 800 ml of M9 medium, and the culture was further continued at 37° C. for 24 hours. The cells were collected from the broth by centrifugation and suspended in 12.5 ml of 20 mM Tris and 30 mM of NaCl. 1 mg/ml of lysozyme and 0.5 MEDTA were added to the obtained suspension, and they were left to stand at 4° C. for 1 hour. Then the cells were crushed by ultrasonication for 20 minutes. The protein inclusion bodies containing denatured MTG were collected by centrifugation. The protein inclusion bodies were thoroughly suspended in a small amount of water, and then, the reagents and water were added to the obtained suspension to a final concentration of 8 M urea·20 mM sodium phosphate·1 mM MEDTA·20 mM DTT/5 ml to dissolve the protein inclusion bodies.

[0139] After incubating at 37° C. for 2 hours, pH was lowered to 4 and the mixture was centrifuged to remove the precipitant. The solution of solubilized MTG was slowly dropped into 250 ml of 20 mM sodium acetate (pH 4.0) 2mM DTT, and the mixture was left to stand for 2 hours. After 2 hours, pH was elevated to 5, and the insoluble matter was removed by centrifugation. pH was elevated to 6 to. complete the refolding. The whole refolding procedure was carried out in a cold room at 4° C.

[0140] After replacing 250 ml of the solution with 20 mM sodium acetate (pH 5.8) with Sephadex G25(M) (Pharmacia), a protein fraction (55 ml) was recovered, which was eluted from an ion exchange resin (CM Sepharose FF) (Pharmacia) by the linear concentration gradient of 0 to 0.4 M of sodium chloride in 20 mM sodium acetate (pH 5.8). Again the solvent was replaced with 20 mM sodium phosphate (pH 6.0) with G25(M).

[0141] When the subsequent amino acid of N-terminal Met is Ser, since N-terminal Met will be removed by methionine aminopeptidase of Escherichia coli, the codon corresponding to Ser may be positioned after the initiation codon to prepare the Ser-type mutant having Ser at the N-terminal.

[0142] (2) Preparation of Mutant by Replacing Ser 2

[0143] For producing three mutant MTGs (S2Y, S2R and S2D) by Escherichia coli, MTG expression plasmid pGEMMTG3 (encoding the amino acid sequence in SEQ ID No: 4 and containing the base sequence of SEQ ID No: 3) was constructed and used as a template for the site-specific mutagenesis using PCR. pGEMMTG3 is a plasmid highly expressing MTG by T7 promoter inducibly, and which was constructed by removing T7 gene 10 protein gene from plasmid pGEM15TG(Xa) expressing the fusion protein of amino-terminal side region of T7 gene 10 protein and MTG [Biosci. Biotech. Biochem. Vol. 61 (5), pp. 830-835 (1997)] and introducing a synthetic gene designed so that a high expression is possible in Escherichia coli as described in JP-Kokai No. 11-75876.

[0144] The procedures for constructing pGEMMTG3 are described below. Firstly, primer pGEMTGF01 (SEQ ID No: 11) which was designed so that 42 residues from T7 gene 10 protein were deleted and which had PstI cleavage site at its 5′ terminal and primer pGEMTGR01 (SEQ ID No: 12) having EcoRI cleavage site in MTG gene were synthesized. PCR was carried out by using these primers and pGEM15TG(Xa) as a template, and the amplified fragments were cloned into pUC19 (Takara Shuzo Co., Ltd.). 25 cycles of PCR were carried out with the conditions of 96° C. for 30 sec, 50° C. for 15 sec and 60° C. for 1 minute. The nucleotide sequence of the insert-containing plasmid was analyzed and the plasmid having the intended correct sequence was named “pUCTGN”. Because pGEM15TG(Xa) had two Ndel cleavage sites, Ndel/EcoRI fragment of pUCTGN was replaced by Ndel/EcoRI fragment of pGEM15TG(Xa) through multiple steps as described below. Namely, the small fragment obtained by cleaving pGEM15TG(Xa) with SmaI and EcoRI was subcloned into pBluescript (Toyobo Co., Ltd.) vector which had been cleaved by SmaI and EcoRI to obtain “pBS15TG(SE)”. Then the fragment containing TG gene obtained by cleaving pUCTGN with Ndel and EcoRI was ligated to the segment obtained by cleaving pBS15TG(SE) with Ndel and EcoRI, which did not contain TG gene, to obtain “pBSTG(SE)”. Further, the large fragment obtained by cleaving pGEM15TG(Xa) with SmaI and EcoRI was ligated to the fragment containing TG gene obtained by cleaving pBSTG(SE) with Ndel and EcoRI to obtain “pGEMTG”. Finally, the MTG gene segment (PvuII-HindIII cleavage fragment) of pGEMTG was replaced with MTG gene (PvuII-HindIII cleavage fragment) of pUCTRPMTG-02(+) (described in JP-Kokai No. 11-75876) to construct pGEMMTG3.

[0145] The mutation was introduced using quick exchange (registered trade mark) site-directed mutagenesis kit (Stratagene) with a primer (FIG. 5, SEQ ID NOS: 13-18) corresponding to each mutant enzyme according to the manufacturer's protocol. Namely, FIG. 5 shows the primer sets used in the site-directed mutagenesis for S2Y·S2R·S2D mutant. Escherichia coli JM109 (Takara Shuzo Co., Ltd.) was transformed by using the PCR product. The transformed cells were plated on L agar medium plates containing 100 μl/ml of ampicillin and incubated at 37° C. for 16 hours. Colonies thus formed were taken and cultured in L-medium containing 100 μl/ml of ampicillin by shaking overnight. The cells were recovered from the broth by centrifugation, and the plasmid was extracted by using Automatic DNA Isolation System PI-50 (KURABO) according to the manufacturer's protocol. The nucleotide sequence encoding each mutant MTG was confirmed by DNA sequencing. The plasmid was transformed into Escherichia coli BL21 (Promega).

[0146]Escherichia coli BL21(DE3) into which the mutant MTG gene had been introduced was inoculated into 50 ml of L medium (10 g/L of Tryptone, 5 g/L of yeast extract, 15 g/L of NaCl and 1 g/L of glucose, pH 7.2) containing 200 μg/ml of ampicillin, and pre-cultured at 37° C. for 6 hours. 8 ml of the pre-culture was transferred into M9 Casamino acid medium (8 g/L of casamino acid, 5 g/L of ammonium chloride, 0.2 g/L of yeast extract, 2 mg/L of vitamin B, hydrochloride, 14.5 mg/L of calcium chloride dihydrate, 15.1 g/L of disodium hydrogen phosphate 12 hydrate, 3 g/L of potassium dihydrogen phosphate, 0.5 g/L of magnesium sulfate heptahydrate and 5 g/L of glucose, pH 7.0) containing 200 μg/ml of ampicillin, and further cultured at 37° C. for 4 hours. When OD₆₆₀ reached to about 0.6, 0.8 ml of 1 M IPTG was added to the culture, and the culture was further continued for 14 hours at 37° C. The treatment of the cells after the culture, the solubilization of the protein inclusion body, the protein refolding and the purification of the protein were carried out as described in (1).

[0147] (3) Preparation of Mutants in Which the Acidic Amino Acid Residues are Modified

[0148] MTG expression plasmid pGEMMTG3 was used as the template for the site-specific mutagenesis and synthetic DNA (SEQ ID NOS: 19-22 in FIG. 6) corresponding to each mutant enzyme was used as the primer. The construction of the mutant plasmid, the culture, the treatment of the cells, the solubilization of the protein inclusion body, the protein refolding and the purification of protein were carried out as described in Example 4 (2).

[0149] (4) Preparation of the Mutants, Sg4 and Sg7 in Which the Loops Were Removed

[0150] For Sg4 genes, the genes corresponding to residue Nos. 1-240 and 253-331 was excised, and for Sg7 gene, the genes corresponding to residue Nos. 1-277 and 288-331 was excised, and then the two fragments were ligated together, respectively. Methods for constructing the Sg4 expression plasmid and the Sg7 expression plasmid are described below. PCR was carried out by using the primer (SEQ ID NO: 24) having SmaI cleavage site at the 5′-terminal and the primers (SEQ ID NOS: 25 and 26) containing a fragment encoding 3 glycine residues at the 3′-terminal, and pGEMMTG3 as the template. Similarly, PCR was carried out by using the primers (SEQ ID NOS: 27 and 28) containing a fragment encoding 3 glycine residues at the 5′-terminal and the primer (SEQ ID NO: 29) having HindIII cleavage site at the 3′-terminal, and pGEMMTG3 as the template. PCR was then carried out by using the primer (SEQ ID NO: 24) having SmaI cleavage site at the 5′-terminal and the primer (SEQ ID NO: 29) having HindIII cleavage site at the 3′-terminal together with the two amplified DNA fragments as the templates to obtain amplified Sg4 gene and Sg7 gene. For Sg7 gene, the fragment was cleaved by SmaI and HindIII, and cloned in pGEMMTG3 between SmaI site and HindIII site. On the other hand, for Sg7, the fragment was cleaved by EcoRI and HindIII, and cloned in pGEMMTG3 between EcoRI site and HindIII site. Further, the nucleotide sequences of the inserts of the obtained plasmids were confirmed to complete the construction of the Sg4 expression plasmid and the Sg7 expression plasmid.

[0151] The methods of the culture, cell treatment, solubilization of the protein inclusion body, protein refolding and protein purification were carried out as described in Example 4 (2).

Example 5

[0152] Comparison of Substrate Specificity

[0153] For comparing the substrate specificity, ovalbumin was employed as the substrate protein. Ovalbumin has 12 glutamine residues, and the reactivity to each of them was traced by NMR. For utilizing the protein in the fields of foods, medicines, etc., for example, the crosslinking rate of the protein molecules should be increased. Therefore, for some of the mutants, the time required for gelling casein in the respective cases was also compared with each other.

[0154] (1) Analysis of Substrate Specificity using NMR

[0155] (1-1) Comparison Between Wild Type and Ser Type

[0156] Ovalbumin, ¹⁵NH₄Cl and transglutaminase were mixed together so that the final concentration is 100 mg/ml for ovalbumin, 200 mM for ¹⁵NH₄Cl and 1 μM for transglutaminase. The first ¹H-¹⁵N HSQC determination was carried out 20 minutes after, and then the ¹H-¹⁵N HSQC determination was repeated 13 times at an interval of 2 hours and 15 minutes. An example of ¹H-¹⁵N HSQC obtained after reacting the wild type for 3 hours is shown in FIG. 7. For both of the wild type and Ser type, the signal peak intensity (longitudinal axis) shown as “a” in FIG. 7 was plotted against the reaction time (hour; horizontal axis) (FIG. 8). As a result, it was found that the glutamine residue generating the signal indicated as “a” in FIG. 7 was more rapidly ¹⁵N-labeled when it was reacted with the Ser-type rather than when reacted with the wild type.

[0157] (1-2) Comparison of Wild Type with S2Y·S2R·S2D mutant

[0158] Ovalbumin, ¹⁵NH₄Cl and transglutaminase were mixed together so that the final concentration is 100 mg/ml for ovalbumin, 200 mM for ¹⁵NH₄Cl and 1 μM for transglutaminase. 20 minutes after, the first ¹H-¹⁵N HSQC determination was carried out and then the ¹H-¹⁵N HSQC determination was repeated 13 times at an interval of 2 hours and 15 minutes. The peak intensity (longitudinal axis) of the spectra obtained by reacting the wild type, S2Y or S2D on the glutamine residue giving the signal “a” in FIG. 7, was plotted, respectively (FIG. 9). As a result, it was found that as for the glutamine residue generating the signal “a” in FIG. 7, the order of the ¹⁵N-labeling rate was S2Y<S2D<wild type<S2R.

[0159] (1-3) Comparison with Mutants Having Modified Acidic Amino Acid Residues

[0160] Ovalbumin, ¹⁵NH₄Cl and transglutaminase were mixed together so that the final concentration is 100 mg/ml for ovalbumin, 200 mM for ¹⁵NH₄Cl and 1 μM for transglutaminase. 20 minutes after, the first ¹H-¹⁵N HSQC determination was carried out and then the ¹H-¹⁵N HSQC determination was repeated 13 times at an interval of 2 hours and 15 minutes. The peak intensity (longitudinal axis) of the spectra obtained by reacting wild type, Ser type, S2R, del 1-2 or del 1-3 to the glutamine residue giving the signal “a” in FIG. 7 was plotted (FIG. 8) respectively in the same manner as that of Example 5. As a result, it was found that the reactivity of each of del 1-2 and del 1-3 was improved as compared with that of the wild type. Furthermore, it can be understood that the reactivity of del 1-3 was even much higher than that of Ser type or S2R.

[0161] Thus, the cross-linking of proteins can be carried out by using smaller amount of transglutaminase than the amount used in the prior art by generating a mutant transglutaminase having modified substrate specificity by reducing the negative charge through modifying acidic amino acid residues or converting amino acid residues adjacent to those acidic amino acid residues into basic ones, which leads the remarkable reduction of the costs.

[0162] (1-4) Comparison with Mutants in Which the Loop Region is Removed

[0163] Ovalbumin, ¹⁵NH₄Cl and transglutaminase were mixed together so that the final concentration is 100 mg/ml for ovalbumin, 200 mM for ¹⁵NH₄Cl and 1 μM for transglutaminase. 6 hours after, the ¹H-¹⁵N HSQC determination was carried out to find that only 3 groups and 2 groups of the signals were found when Sg4 and Sg7 were used, respectively, while 6 groups of the signals were found when the wild type was used (FIG. 10). It was known from the transglutaminase-free experiments that among the signals observed in FIG. 10, unnumbered ones are not from the glutamine residues labeled by the transglutaminase reaction. As for the spectrum of ovalbumin labeled using Sg7, the figure extracted from a lower level is shown because the reactivity of Sg7 was reduced to lower the labeling efficiency. It is to be noted that, for this reason, unnumbered signals were slightly emphasized.

[0164] Only glutamine residues corresponding to (1), (3) and (4) were labeled using Sg4, while those corresponding to (1) to (6) were labeled with the wild type. Therefore, it was shown that when Sg4 was used, the number of glutamine residues which could serve as the substrates decreased, namely its substrate specificity was higher, compared with the case when the wild type was used. It was also shown that with Sg7, glutamine residues only corresponding to (1) and (3) can be the substrate, although the reactivity was reduced. It was considered that the binding area for the substrate was reduced by removing the loop, and that the kinds of the substrates with which the mutant could be reacted were reduced. As the results, it was suggested that the substrate specificity of MTG could be modified by preparing mutants thereof based on the three-dimensional structure. By using the transglutaminase having modified substrate specificity, the application of transglutaminase to subjects different from previously described may be possible, and the new development in industries is expected.

[0165] (2) Comparison of the Time of Gelatinization of Casein

[0166] 57.6 μg of wild, Ser type, S2Y, S2R or S2D mutant was respectively added to 2.5 g of 8% (w/w) casein solution, and the time required for the gelatination of casein in each case was compared with each other. The reaction temperature was 40° C. The time required for the gelation was 90 minutes for wile type, 85 minutes for Ser type, 100 minutes for S2Y mutant, 85 minutes for S2R mutant and 95 minutes for S2D mutant. Thus, it was shown that the gelatinization proceeded in a short period for Ser type or S2R mutant, and the gelatination slowly proceeded for S2Y and S2D.

[0167] According to the present invention, the improvement of MTG on the basis of the three-dimensional structure is facilitated. In particular, transglutaminase having an improved reactivity on the substrate can be provided by the present invention. Further, new products and novel techniques can be provided with the transglutaminase having the improved reactivity on the substrate.

1 29 1 993 DNA Streptoverticillium mobaraense CDS (1)..(993) 1 gac tcc gac gac agg gtc acc cct ccc gcc gag ccg ctc gac agg atg 48 Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met 1 5 10 15 ccc gac ccg tac cgt ccc tcg tac ggc agg gcc gag acg gtc gtc aac 96 Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn 20 25 30 aac tac ata cgc aag tgg cag cag gtc tac agc cac cgc gac ggc agg 144 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg 35 40 45 aag cag cag atg acc gag gag cag cgg gag tgg ctg tcc tac ggc tgc 192 Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys 50 55 60 gtc ggt gtc acc tgg gtc aat tcg ggt cag tac ccg acg aac aga ctg 240 Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80 gcc ttc gcg tcc ttc gac gag gac agg ttc aag aac gag ctg aag aac 288 Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85 90 95 ggc agg ccc cgg tcc ggc gag acg cgg gcg gag ttc gag ggc cgc gtc 336 Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val 100 105 110 gcg aag gag agc ttc gac gag gag aag ggc ttc cag cgg gcg cgt gag 384 Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu 115 120 125 gtg gcg tcc gtc atg aac agg gcc ctg gag aac gcc cac gac gag agc 432 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser 130 135 140 gct tac ctc gac aac ctc aag aag gaa ctg gcg aac ggc aac gac gcc 480 Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala 145 150 155 160 ctg cgc aac gag gac gcc cgt tcc ccg ttc tac tcg gcg ctg cgg aac 528 Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn 165 170 175 acg ccg tcc ttc aag gag cgg aac gga ggc aat cac gac ccg tcc agg 576 Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg 180 185 190 atg aag gcc gtc atc tac tcg aag cac ttc tgg agc ggc cag gac cgg 624 Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205 tcg agt tcg gcc gac aag agg aag tac ggc gac ccg gac gcc ttc cgc 672 Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 ccc gcc ccg ggc acc ggc ctg gtc gac atg tcg agg gac agg aac att 720 Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile 225 230 235 240 ccg cgc agc ccc acc agc ccc ggt gag gga ttc gtc aat ttc gac tac 768 Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr 245 250 255 ggc tgg ttc ggc gcc cag acg gaa gcg gac gcc gac aag acc gtc tgg 816 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp 260 265 270 acc cac gga aat cac tat cac gcg ccc aat ggc agc ctg ggt gcc atg 864 Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met 275 280 285 cat gtc tac gag agc aag ttc cgc aac tgg tcc gag ggt tac tcg gac 912 His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp 290 295 300 ttc gac cgc gga gcc tat gtg atc acc ttc atc ccc aag agc tgg aac 960 Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 acc gcc ccc gac aag gta aag cag ggc tgg ccg 993 Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330 2 331 PRT Streptoverticillium mobaraense 2 Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met 1 5 10 15 Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val Asn 20 25 30 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg 35 40 45 Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys 50 55 60 Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80 Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85 90 95 Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val 100 105 110 Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu 115 120 125 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser 130 135 140 Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala 145 150 155 160 Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn 165 170 175 Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg 180 185 190 Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205 Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile 225 230 235 240 Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr 245 250 255 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp 260 265 270 Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met 275 280 285 His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp 290 295 300 Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330 3 996 DNA Streptoverticillium mobaraense CDS (1)..(996) 3 atg gat tct gac gat cga gtc act cca cca gct gaa cca ctg gat cgt 48 Met Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg -1 1 5 10 15 atg cca gat cca tat cgt cca tct tat ggt cgt gct gaa act gtt gtt 96 Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val 20 25 30 aat aat tat att cgt aaa tgg caa caa gtt tat tct cat cgt gat ggt 144 Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly 35 40 45 cgt aaa caa caa atg act gaa gaa caa cgt gaa tgg ctg tct tat ggt 192 Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly 50 55 60 tgc gtt ggt gtt act tgg gtt aac tct ggt cag tat ccg act aac cgt 240 Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg 65 70 75 ctg gca ttc gct tcc ttc gat gaa gat cgt ttc aag aac gaa ctg aag 288 Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys 80 85 90 95 aac ggt cgt ccg cgt tct ggt gaa act cgt gct gaa ttc gaa ggt cgt 336 Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg 100 105 110 gtt gct aag gaa tcc ttc gat gaa gag aaa ggc ttc cag cgt gct cgt 384 Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg 115 120 125 gaa gtt gct tct gtt atg aac cgt gct cta gag aac gct cat gat gaa 432 Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu 130 135 140 tct gct tac ctg gat aac ctg aag aag gaa ctg gct aac ggt aac gat 480 Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp 145 150 155 gct ctg cgt aac gaa gat gct cgt tct ccg ttc tac tct gct ctg cgt 528 Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg 160 165 170 175 aac act ccg tcc ttc aaa gaa cgt aac ggt ggt aac cat gat ccg tct 576 Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser 180 185 190 cgt atg aaa gct gtt atc tac tct aaa cat ttc tgg tct ggt cag gat 624 Arg Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp 195 200 205 aga tct tct tct gct gat aaa cgt aaa tac ggt gat ccg gat gca ttc 672 Arg Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe 210 215 220 cgt ccg gct ccg ggt act ggt ctg gta gac atg tct cgt gat cgt aac 720 Arg Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn 225 230 235 atc ccg cgt tct ccg act tct ccg ggt gaa ggc ttc gtt aac ttc gat 768 Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp 240 245 250 255 tac ggt tgg ttc ggt gct cag act gaa gct gat gct gat aag act gta 816 Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val 260 265 270 tgg acc cat ggt aac cat tac cat gct ccg aac ggt tct ctg ggt gct 864 Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala 275 280 285 atg cat gta tac gaa tct aaa ttc cgt aac tgg tct gaa ggt tac tct 912 Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser 290 295 300 gac ttc gat cgt ggt gct tac gtt atc acc ttc att ccg aaa tct tgg 960 Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp 305 310 315 aac act gct ccg gac aaa gtt aaa cag ggt tgg ccg 996 Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 320 325 330 4 332 PRT Streptoverticillium mobaraense 4 Met Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg -1 1 5 10 15 Met Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr Val Val 20 25 30 Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly 35 40 45 Arg Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly 50 55 60 Cys Val Gly Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg 65 70 75 Leu Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys 80 85 90 95 Asn Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg 100 105 110 Val Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg 115 120 125 Glu Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu 130 135 140 Ser Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp 145 150 155 Ala Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg 160 165 170 175 Asn Thr Pro Ser Phe Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser 180 185 190 Arg Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp 195 200 205 Arg Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe 210 215 220 Arg Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn 225 230 235 Ile Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp 240 245 250 255 Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val 260 265 270 Trp Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala 275 280 285 Met His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Glu Gly Tyr Ser 290 295 300 Asp Phe Asp Arg Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp 305 310 315 Asn Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 320 325 330 5 990 DNA Streptoverticillium cinnamoneum CDS (1)..(990) 5 tcc cgg gcc ccc tcc gat gac cgg gaa act cct ccc gcc gag ccg ctc 48 Ser Arg Ala Pro Ser Asp Asp Arg Glu Thr Pro Pro Ala Glu Pro Leu 1 5 10 15 gac agg atg cct gag gcg tac cgg gcc tac gga ggc agg gcc act acg 96 Asp Arg Met Pro Glu Ala Tyr Arg Ala Tyr Gly Gly Arg Ala Thr Thr 20 25 30 gtc gtc aac aac tac ata cgc aag tgg cag cag gtc tac agt cac cgc 144 Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg 35 40 45 gac gga aag aaa cag caa atg acc gaa gag cag cga gaa aag ctg tcc 192 Asp Gly Lys Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Lys Leu Ser 50 55 60 tac ggt tgc gtt ggc gtc acc tgg gtc aac tcg ggc ccc tac ccg acg 240 Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Pro Tyr Pro Thr 65 70 75 80 aac aga ttg gcg ttc ggt ccc ttc gac gag aac aag tac aag aac gac 288 Asn Arg Leu Ala Phe Gly Pro Phe Asp Glu Asn Lys Tyr Lys Asn Asp 85 90 95 ctg aag aac acc agc ccc cga ccc gat gaa acg cgg gcg gag ttc gag 336 Leu Lys Asn Thr Ser Pro Arg Pro Asp Glu Thr Arg Ala Glu Phe Glu 100 105 110 ggt cgc atc aag ggc agt ttc gac gag ggg aag ggt ttc aag cgg gcg 384 Gly Arg Ile Lys Gly Ser Phe Asp Glu Gly Lys Gly Phe Lys Arg Ala 115 120 125 cgt gat gtg gcg tcc gtc atg aac aag gcc cgg gaa aat gcc cac gac 432 Arg Asp Val Ala Ser Val Met Asn Lys Ala Arg Glu Asn Ala His Asp 130 135 140 gag ggg act tac atc aac aac ctc aag acg gag ctc acg aac aac aat 480 Glu Gly Thr Tyr Ile Asn Asn Leu Lys Thr Glu Leu Thr Asn Asn Asn 145 150 155 160 gcg ctc cgc cgc gag gac agc cgc tcg aac ttc tac tcg gcg ctg agg 528 Ala Leu Arg Arg Glu Asp Ser Arg Ser Asn Phe Tyr Ser Ala Leu Arg 165 170 175 aac aca ccg tcc ttc aag gaa agg gac ggc ggc aac tac gac ccg tcc 576 Asn Thr Pro Ser Phe Lys Glu Arg Asp Gly Gly Asn Tyr Asp Pro Ser 180 185 190 aag atg aag gcg gtg atc tac tcg aag cac ttc tgg agc ggg cag gac 624 Lys Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp 195 200 205 cag cgg ggc tcc tcc gac aag agg aag tac ggc gac ccg gaa gcc ttc 672 Gln Arg Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe 210 215 220 cgc ccc gta cca ggt acc ggc ctg gtc gac atg tcg aag gac aga agc 720 Arg Pro Val Pro Gly Thr Gly Leu Val Asp Met Ser Lys Asp Arg Ser 225 230 235 240 att ccg cgc agt ccg gcc aag ccc ggc gaa ggt tgg gtc aat ttc gac 768 Ile Pro Arg Ser Pro Ala Lys Pro Gly Glu Gly Trp Val Asn Phe Asp 245 250 255 tac ggt tgg ttc ggg gct caa aca gaa gcg gat cgc gac aaa acc aca 816 Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Arg Asp Lys Thr Thr 260 265 270 tgg acc cac ggc gac cac tac cac gcg ccc aat agc gac ctg ggc ccc 864 Trp Thr His Gly Asp His Tyr His Ala Pro Asn Ser Asp Leu Gly Pro 275 280 285 atg cac gta cac gag agc aag ttc cgg aag tgg tct gcc ggg tac gcg 912 Met His Val His Glu Ser Lys Phe Arg Lys Trp Ser Ala Gly Tyr Ala 290 295 300 gac ttc ggc gcc tac gtg atc acg ttc ata ccc aag agc tgg aac acc 960 Asp Phe Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr 305 310 315 320 gcc ccc gcc aag gtg gag caa ggc tgg ccg 990 Ala Pro Ala Lys Val Glu Gln Gly Trp Pro 325 330 6 330 PRT Streptoverticillium cinnamoneum 6 Ser Arg Ala Pro Ser Asp Asp Arg Glu Thr Pro Pro Ala Glu Pro Leu 1 5 10 15 Asp Arg Met Pro Glu Ala Tyr Arg Ala Tyr Gly Gly Arg Ala Thr Thr 20 25 30 Val Val Asn Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg 35 40 45 Asp Gly Lys Lys Gln Gln Met Thr Glu Glu Gln Arg Glu Lys Leu Ser 50 55 60 Tyr Gly Cys Val Gly Val Thr Trp Val Asn Ser Gly Pro Tyr Pro Thr 65 70 75 80 Asn Arg Leu Ala Phe Gly Pro Phe Asp Glu Asn Lys Tyr Lys Asn Asp 85 90 95 Leu Lys Asn Thr Ser Pro Arg Pro Asp Glu Thr Arg Ala Glu Phe Glu 100 105 110 Gly Arg Ile Lys Gly Ser Phe Asp Glu Gly Lys Gly Phe Lys Arg Ala 115 120 125 Arg Asp Val Ala Ser Val Met Asn Lys Ala Arg Glu Asn Ala His Asp 130 135 140 Glu Gly Thr Tyr Ile Asn Asn Leu Lys Thr Glu Leu Thr Asn Asn Asn 145 150 155 160 Ala Leu Arg Arg Glu Asp Ser Arg Ser Asn Phe Tyr Ser Ala Leu Arg 165 170 175 Asn Thr Pro Ser Phe Lys Glu Arg Asp Gly Gly Asn Tyr Asp Pro Ser 180 185 190 Lys Met Lys Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp 195 200 205 Gln Arg Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe 210 215 220 Arg Pro Val Pro Gly Thr Gly Leu Val Asp Met Ser Lys Asp Arg Ser 225 230 235 240 Ile Pro Arg Ser Pro Ala Lys Pro Gly Glu Gly Trp Val Asn Phe Asp 245 250 255 Tyr Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Arg Asp Lys Thr Thr 260 265 270 Trp Thr His Gly Asp His Tyr His Ala Pro Asn Ser Asp Leu Gly Pro 275 280 285 Met His Val His Glu Ser Lys Phe Arg Lys Trp Ser Ala Gly Tyr Ala 290 295 300 Asp Phe Gly Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn Thr 305 310 315 320 Ala Pro Ala Lys Val Glu Gln Gly Trp Pro 325 330 7 993 DNA Streptomyces lydicus CDS (1)..(993) 7 gca gcc gac gaa agg gtc acc cct ccc gcc gag ccg ctc aac cgg atg 48 Ala Ala Asp Glu Arg Val Thr Pro Pro Ala Glu Pro Leu Asn Arg Met 1 5 10 15 cct gac gcg tac cgg gcc tac gga ggt agg gcc act acg gtc gtc aac 96 Pro Asp Ala Tyr Arg Ala Tyr Gly Gly Arg Ala Thr Thr Val Val Asn 20 25 30 aac tac ata cgc aag tgg cag cag gtc tac agt cac cgc gac ggc atc 144 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Ile 35 40 45 caa cag caa atg acc gaa gag cag cga gaa aag ctg tcc tac ggc tgc 192 Gln Gln Gln Met Thr Glu Glu Gln Arg Glu Lys Leu Ser Tyr Gly Cys 50 55 60 gtc ggc atc acc tgg gtc aat tcg ggc ccc tac ccg acg aat aaa ttg 240 Val Gly Ile Thr Trp Val Asn Ser Gly Pro Tyr Pro Thr Asn Lys Leu 65 70 75 80 gcg ttc gcg ttc ttc gac gag aac aag tac aag agt gac ctg gaa aac 288 Ala Phe Ala Phe Phe Asp Glu Asn Lys Tyr Lys Ser Asp Leu Glu Asn 85 90 95 agc agg cca cgc ccc aat gag acg caa gcc gag ttt gag ggg cgc atc 336 Ser Arg Pro Arg Pro Asn Glu Thr Gln Ala Glu Phe Glu Gly Arg Ile 100 105 110 gtc aag gac agt ttc gac gag ggg aag ggt ttc aag cgg gcg cgt gat 384 Val Lys Asp Ser Phe Asp Glu Gly Lys Gly Phe Lys Arg Ala Arg Asp 115 120 125 gtg gcg tcc gtc atg aac aag gcc ctg gat agt gcg cac gac gag ggg 432 Val Ala Ser Val Met Asn Lys Ala Leu Asp Ser Ala His Asp Glu Gly 130 135 140 act tac atc gac aac ctc aag acg gag ctc gcg aac aaa aat gac gct 480 Thr Tyr Ile Asp Asn Leu Lys Thr Glu Leu Ala Asn Lys Asn Asp Ala 145 150 155 160 ctg cgc tac gag gac ggt cgc tcg aac ttt tac tcg gcg ctg agg aat 528 Leu Arg Tyr Glu Asp Gly Arg Ser Asn Phe Tyr Ser Ala Leu Arg Asn 165 170 175 acg ccg tcc ttc aag gaa agg gat gga ggt aac tac gac cca tcc aag 576 Thr Pro Ser Phe Lys Glu Arg Asp Gly Gly Asn Tyr Asp Pro Ser Lys 180 185 190 atg aag gcg gtg gtc tac tcg aaa cac ttc tgg agc ggg cag gac cag 624 Met Lys Ala Val Val Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Gln 195 200 205 cgg ggc tcc tct gac aag agg aag tac ggc gac ccg gat gcc ttc cgc 672 Arg Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 ccc gac cag ggc aca ggc ctg gta gac atg tcg aag gac agg aat att 720 Pro Asp Gln Gly Thr Gly Leu Val Asp Met Ser Lys Asp Arg Asn Ile 225 230 235 240 ccg cgc agt ccc gcc caa cct ggc gaa agt tgg gtc aat ttc gac tac 768 Pro Arg Ser Pro Ala Gln Pro Gly Glu Ser Trp Val Asn Phe Asp Tyr 245 250 255 ggc tgg ttt ggg gct cag acg gaa tcg gac gcc gac aaa acc ata tgg 816 Gly Trp Phe Gly Ala Gln Thr Glu Ser Asp Ala Asp Lys Thr Ile Trp 260 265 270 acc cac gcc aac cac tat cac gcg ccc aac ggc ggc ctg ggc ccc atg 864 Thr His Ala Asn His Tyr His Ala Pro Asn Gly Gly Leu Gly Pro Met 275 280 285 aac gta tat gag agc aag ttc cgg aac tgg tct gcc ggg tac gcg gat 912 Asn Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Ala Gly Tyr Ala Asp 290 295 300 ttc gac cgc gga acc tac gtc atc acg ttc ata ccc aag agc tgg aac 960 Phe Asp Arg Gly Thr Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 acc gcc ccc gcc gag gta aag cag ggc tgg tcg 993 Thr Ala Pro Ala Glu Val Lys Gln Gly Trp Ser 325 330 8 331 PRT Streptomyces lydicus 8 Ala Ala Asp Glu Arg Val Thr Pro Pro Ala Glu Pro Leu Asn Arg Met 1 5 10 15 Pro Asp Ala Tyr Arg Ala Tyr Gly Gly Arg Ala Thr Thr Val Val Asn 20 25 30 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Ile 35 40 45 Gln Gln Gln Met Thr Glu Glu Gln Arg Glu Lys Leu Ser Tyr Gly Cys 50 55 60 Val Gly Ile Thr Trp Val Asn Ser Gly Pro Tyr Pro Thr Asn Lys Leu 65 70 75 80 Ala Phe Ala Phe Phe Asp Glu Asn Lys Tyr Lys Ser Asp Leu Glu Asn 85 90 95 Ser Arg Pro Arg Pro Asn Glu Thr Gln Ala Glu Phe Glu Gly Arg Ile 100 105 110 Val Lys Asp Ser Phe Asp Glu Gly Lys Gly Phe Lys Arg Ala Arg Asp 115 120 125 Val Ala Ser Val Met Asn Lys Ala Leu Asp Ser Ala His Asp Glu Gly 130 135 140 Thr Tyr Ile Asp Asn Leu Lys Thr Glu Leu Ala Asn Lys Asn Asp Ala 145 150 155 160 Leu Arg Tyr Glu Asp Gly Arg Ser Asn Phe Tyr Ser Ala Leu Arg Asn 165 170 175 Thr Pro Ser Phe Lys Glu Arg Asp Gly Gly Asn Tyr Asp Pro Ser Lys 180 185 190 Met Lys Ala Val Val Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Gln 195 200 205 Arg Gly Ser Ser Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 Pro Asp Gln Gly Thr Gly Leu Val Asp Met Ser Lys Asp Arg Asn Ile 225 230 235 240 Pro Arg Ser Pro Ala Gln Pro Gly Glu Ser Trp Val Asn Phe Asp Tyr 245 250 255 Gly Trp Phe Gly Ala Gln Thr Glu Ser Asp Ala Asp Lys Thr Ile Trp 260 265 270 Thr His Ala Asn His Tyr His Ala Pro Asn Gly Gly Leu Gly Pro Met 275 280 285 Asn Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser Ala Gly Tyr Ala Asp 290 295 300 Phe Asp Arg Gly Thr Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 Thr Ala Pro Ala Glu Val Lys Gln Gly Trp Ser 325 330 9 42 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 9 cggatccatc gaaggtcgtg attctgacga tcgtgttact cc 42 10 30 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 10 caatttgcga gctcattacg gccaaccctg 30 11 42 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 11 cctgcagaag gagatataca tatggattct gacgatcgag tc 42 12 19 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 12 catcgaaaga ttccttagc 19 13 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 13 ggagatatac atatggatta cgacgatcgt gttactcc 38 14 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 14 ggagatatac atatggatta cgacgatcgt gttactcc 38 15 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 15 ggagatatac atatggatcg tgacgatcgt gttactcc 38 16 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 ggagtaacac gatcgtcacg atccatatgt atatctcc 38 17 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 17 ggagatatac atatggatga cgacgatcgt gttactcc 38 18 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 18 ggagtaacac gatcgtcgtc atccatatgt atatctcc 38 19 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 19 ggagatatac atatggacga tcgtgttact ccaccagc 38 20 38 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 20 gctggtggag taacacgatc gtccatatgt atatctcc 38 21 40 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 21 gaaggagata tacatatgga tcgtgttact ccaccagctg 40 22 40 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 22 cagctggtgg agtaacacga tccatatgta tatctccttc 40 23 21 PRT ARTIFICIAL SEQUENCE SYNTHETIC PEPTIDE 23 Gly Ile Val Glu Gln Cys Cys Ala Ser Val Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 24 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 24 gattcattaa tgcagcccgg gagatc 26 25 50 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 25 ccgatatcga agttaccgcc accgatgtta cgatcacgag acatgtctac 50 26 51 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 26 gattcgtata catgcatacc gccaccatgg ttaccatggg tccatacagt c 51 27 35 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 27 ggtggcggta acttcgatta cggttggttc ggtgc 35 28 35 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 28 ggtggcggta tgcatgtata cgaatctaaa ttccg 35 29 25 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 29 gaatactcaa gctttcatta cggcc 25 

What is claimed is:
 1. A method for designing and preparing a mutant transglutaminase by extrapolating the binding site of a transglutaminase derived from Streptoverticillium mobaraense (MTG) for the substrate on the basis of the three-dimensional structure obtained by X-ray crystal structure analysis and replacing, inserting or deleting amino acid residues positioned at the extrapolated substrate-binding site of the transglutaminase.
 2. A mutant MTG (excluding a mutant MTG wherein Asp¹is deleted) having modified substrate specificity, in which at least one of the following positions (residues positioned within 20 Å from active residue Cys⁶⁴ and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296,-297 and 300-304.
 3. A mutant transglutaminase having transglutaminase activity and modified substrate specificity, wherein when the alignment of the sequence of said mutant transglutaminase with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out, at least one of the positions corresponding to the following positions (residues positioned within 20 Å from active residue Cys⁶⁴ and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequence according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296-297 and 300-304.
 4. A mutant transglutaminase having transglutaminase activity and modified substrate specificity, wherein when the alignment of the three-dimensional structure with the MTG amino acid sequence according to SEQ ID NO: 2 is carried out by treading method, at least one of the positions corresponding to the following positions (residues positioned within 20 Å from active residue Cys⁶⁴ and exposed on the surface of the extrapolated binding site for acyl acceptor) in the MTG amino acid sequences according to SEQ ID No: 2 is modified: 1-5, 26, 28, 58-59, 62, 69, 74-75, 77, 79, 235-236, 238-244, 248-250, 252-254, 277-278, 282-287, 289, 291, 296-297 and 300-304.
 5. A mutant MTG (excluding a mutant MTG from which Asp¹ is deleted) or mutant transglutaminase having modified substrate specificity, wherein a negative charge is reduced by modifying an acidic amino acid residue or an amino acid residue adjacent to the acidic amino acid located at the position described in any one of claims 2 to 4
 6. A gene encoding the mutant MTG or the mutant transglutaminase according to any one of claims 2 to
 4. 7. A recombinant DNA containing the gene according to claim
 6. 8. A microorganisms containing the recombinant DNA according to claim
 7. 9. A method for producing a mutant MTG or a mutant transglutaminase, which comprises culturing the microorganisms according to claim 8 and collecting the mutant MTG or the mutant transglutaminase.
 10. A monoclinic MTG crystals belonging to P2₁ space group. 